It is well known that the incorporation or substitution of a D-Tryptophan residue into a biologically active peptide chain enhances the activity of that chain. Furthermore, such incorporation or substitution will prolong the biological activity. The prolonged and enhanced effectiveness of such peptides probably relates the increased resistance to degradation by peptidases.
Examples of D-Tryptophan containing peptides are the LHRH agonists as described by D. H. Coy et al., Journal of Medical Chemistry, volume 19, page 423 (1976), W. Koenig et al., Peptide Chemistry (1987), T. Shiba and S. Sakakibara (eds.), Osaka, Protein Research Foundation, Osaka (1988), page 591, B. J. A. Furr et al., Journal of Endocrinol. Invest., volume 11, page 535 (1988). Examples of D-Tryptophan containing somastostatin analogs, such as the peptides octreotide and vapreotide are disclosed by R. Deghenghi, Biomedicine and Pharmacotherapy, volume 42, page 585 (1988). Another example of a D-Tryptophan containing peptide are the synthetic antagonists of Substance P as disclosed by D. Regoli et al., European Journal of Pharmacology, volume 99, page 193, (1984), and GHRP-6 described by C. Y. Bowers et al., Endocrinology, volume 114, page 1537, (1984).
Peptides containing Tryptophan have been subject to degradation due to the "Kynurenine pathway". In this pathway, the enzyme Tryptophan pyrrolase (i.e., indolamine 2,3-dioxygenase) degrades the pyrrole ring of Tryptophan. Kynurenine and other breakdown products are generated by this degradation. Some of the breakdown products have been shown to be toxic when present in elevated concentrations as reported by R. M. Silver et al., The New England Journal of Medicine, volume 322, page 874, (1990).
D-Tryptophan containing peptides are subject to degradation by oxygen and other reactive radicals as reported by R. Geiger and W. Koenig, "The Peptides," Academic Press, volume 3, page 82, New York (1981). The D-Tryptophan in the peptide chain may react with active or activated groups when peptides are formulated in certain controlled delivery pharmaceutical compositions, such as those based on polylactic/polyglycolic acid polymers. Such degradation is thought to be facilitated by either heat or by the presence of catalysts. It is also possible that radiolysis products formed during ionizing sterilization of these pharmaceutical compositions may facilitate the breakdown of D-Tryptophan. Clearly, the breakdown of D-Tryptophan, and the concomitant breakdown of the pharmaceutical compound containing D-Tryptophan is an undesirable effect.
Yabe et al., Synthesis and Biological Activity of LHRH Analogs Substituted by Alkyl Tryptophans at Position 3, Chem. Pharm. Bul. 27 (8) pp. 1907-1911 (1979) discloses seven analogs of LHRH in which the Tryptophan residue at position 3 was replaced by various L-methyl Tryptophans and L-ethyl Tryptophans. However, each analog tested exhibited reduced hormonal activity compared to synthetic LHRH.
What is needed is a derivative of D-Tryptophan which retains the prolonged and increased biological activity discussed above, while resisting degradation by indolamine dioxygenase, oxygen or other reactive radicals. It is of course essential that such a derivative of D-Tryptophan would maintain biological activity as compared to D-Tryptophan containing bioactive peptides.
One use of such active peptides is for releasing growth hormone ("GH"). If sufficiently high GH levels are achieved in mammals after the administration of compounds capable of inducing such release, growth can be accelerated, muscular mass can be increased and production of milk can be enhanced. It is known that the increase of growth hormone levels in mammals can be achieved by administering growth hormone release agents, such as, for example, growth hormone release hormones (GHRH).
The increase of growth hormone levels in mammals can also be obtained by administering growth hormone release peptides. See, for example, the following U.S. Pat. Nos. 4,223,019, 4,223,020, 4,223,021, 4,224,316, 4,226,857, 4,228,155, 4,228,156, 4,228,157, 4,228,158, 4,410,512, 4,410,513, 4,411,890 and 4,839,344. Many of the peptides described in these patents have complex structures, are difficult to synthesize, purify and/or formulate into convenient dosage forms. Additionally, some of these have in vitro activity, but do not exhibit in vivo activity. Further, some of these peptides are not active when administered orally.
One of the more studied growth hormone release peptides is GHRP-6 (C. Y. Bowers et al., Endocrinology 114:1537 (1984) and has the formula His-D-Trp-Ala-Trp-D-Phe-Lys-NH.sub.2. GHRP-6 releases growth hormone both in vitro and in vivo and is orally active in animals, including humans. Its molecular mechanism has been studied, as well as the molecular mechanism of its analogue heptapeptide GHRP-1 (Cheng et al., Endocrinology 124:2791 (1989); M. S. Akman et al., Endocrinology 132:1286 (1993)). It was found that contrary to natural GHRH, GHRP-1 and GHRP-6 act through different receptors for the release of GH and also via a different mechanism, which is independent from cAMP and which operates through other intracellular pathways, such as through the mobilization of calcium supplies and via a proteinkinase C (PKC)-dependent process (L. Bresson-Bepoldin and L. Dufy-Barbe, Cell. Calcium 15, 247 (1994)).
In view of the important effects that growth hormone releasing peptides can have on veterinary and human medicine, there remains a need for growth hormone releasing peptides that are more efficacious than those currently in existence, and as such, can be administered at a lower concentration and at a lower cost with fewer adverse health affects.
Therefore, rather simple, short chain oligopeptides capable of promoting growth hormone release that can be easily and conveniently prepared and that can be easily purified and formulated into a dosage form that can be administered via the oral route are presently desired. In particular, those oligopeptides exhibiting in vivo activity when administered orally are sought.
The terms "biological effect" or "pharmacological effect" as used in the present disclosure refer to the qualitative effect that a bioactive peptide has upon living tissue. As an example, LHRH, luteinizing hormone releasing hormone, has the biological effect of causing cells of the anterior pituitary gland to release luteinizing hormone. In contrast, the term "potency" is used in its conventional sense to refer to the degree and duration of the bioactivity of a given peptide.
Utilizing these terms as defined above, what is needed is a Tryptbphan containing bioactive peptide which is resistant to oxidative degradation and reactive radical attack while maintaining the same biological activity and a similar or greater potency than the presently available analogous peptides provide.